The present invention relates to thermostable glutamate dehydrogenase which is stable in an alkaline solution, and a process for its mass production. This invention also relates to glutamate dehydrogenase which can utilize NAD.sup.+, a more stable coenzyme, as a coenzyme to be involved in a reaction, and an ammonia assay kit using such glutamate dehydrogenase.
Glutamate dehydrogenase (hereinafter referred to as "GLDH") is an enzyme which catalyzes a reaction of the following formula (1):
______________________________________ 2-oxoglutaric acid + NH.sub.3 + NAD(P)H .uparw..dwnarw. (1) glutamic acid + H.sub.2 O + NAD(P).sup.+ ______________________________________
This enzyme is widely distributed among animals, plants and microorganisms. So far, there has been known GLDH of various origins, including bacteria of the genera Proteus, Bacillus and Thermococcus. As an enzyme for applications such as determination of glutamic acid and clinical diagnosis, GLDH has attracted marked attention in recent years in the fields of biochemistry and medicine. The enzymatic activity of GLDH catalyzes the reaction of formula (1) in both directions. In the downward reaction by GLDH, ammonia is used as one of the reactants. Thus, ammonia in a sample produced from an in vivo substance and/or the in vivo substance in a sample can be quantitatively determined by use of this enzyme.
Recent years have seen a tendency toward the replacement of many assay reagent compositions for laboratory examination in a lyophilized powdery form by those in the form of a solution. This replacement is intended to omit the step of converting the powdery assay reagent composition to a solution each time it is to be used, thus reducing the burden on laboratory technicians. In developing a liquid reagent containing GLDH, use of an alkaline solution is required for ensuring stability of NAD(P)H (reduced nicotinamide adenine dinucleotide (phosphate)) which functions as a coenzyme in the reagent. Such use of an alkaline solution, however, has proved defective in that GLDH is deactivated rapidly, and long-term storage of the reagent is difficult. Furthermore, the reagent available is not suitable for long-term storage, as it is susceptible in terms of enzyme stability to accidental temperature changes.
We, the inventors of the present invention, have shown that thermostable GLDH originating from Pyrococcus furiosus has excellent stability under temperature changes during storage, and is stabile in an alkaline solution, and have disclosed that this GLDH can maintain high enzymatic activity in solution for a long period of time (Japanese Patent Application No. 310768/97).
Using conventional methods, however, the production cost of recombinant GLDH is high. There are a number of reasons for this. That is, the expression of recombinant GLDH has required that the expression of a recombinant gene be induced by the use of a drug (isopropyl-.beta.-D(-)-thiogalactopyranoside; IPTG). In addition, the output of GLDH per wet weight of the bacterial cells has been low, and consequently the productivity of culture has also been low. Due to the above-mentioned induction by the drug, moreover, there has been no choice but to obtain a transformant by batch culture. These restrictions have resulted in continuing high manufacturing costs. In addition, thermostable GLDH of the Pyrococcus furiosus origin has been unable to utilize NADH, a more stable coenzyme, with the result that this enzyme has been unsuitable for the production of a reagent kit which is stable during long-term storage.
To overcome the foregoing drawbacks, it has been necessary to search for GLDH which has selectivity for NADH as a coenzyme. There has been a need to develop a process which enables large scale culture by the use of such GLDH, but without the use of a drug for induction of expression. However, no method for mass production of GLDH has hitherto been known, nor has such a preferred enzyme been known. Hence, the aforementioned problems have remained unsolved.